PROJECT 1: Summary/Abstract Identification of tumor-associated mutated target antigens in individual cancer patients can facilitate a novel and potentially effective treatment approach in which an immune response against multiple relevant tumor antigens can be generated using a personalized vaccination approach. While identification of mutated peptide epitope targets in individual patients remains a daunting technical challenge, recent advances in next generation genetic sequencing has provided a strong foundation on which to build these efforts. This approach holds the promise of a more personalized and effective method of activating anti-tumor immunity, without the toxicities associated with many alternate approaches. However, many fundamental questions remain regarding choice of optimal tumor antigens and immunostimulatory adjuvants to use for generating optimal antitumor immunity in cancer patients through vaccination. The specific objective of this proposal is to generate an effective, personalized vaccine approach for treatment of metastatic colorectal cancer (CRC) patients. We will test the hypothesis that a vaccination strategy targeting multiple mutated CRC tumor antigens along with specific combinations of immune adjuvants will be capable of preventing recurrence of minimal residual disease in post-hepatectomy CRC patients. Our Preliminary Data shows that the proposed personalized vaccination strategy is feasible, as several CRC patients have now undergone vaccination in a current clinical trial. In addition, our pre-clinical mouse models have clearly shown that Toll-like receptor ligands, anti-CD40, and checkpoint blockade can be highly effective combinations of adjuvants in vivo. However, it is critical to understand the optimal combination of agents to use for vaccination in order to translate these findings into more effective vaccine strategies for our CRC patients. We thus plan to focus our efforts on the following Specific Aims: Aim 1: Design and immunize 20 post-hepatectomy metastatic CRC patients with personalized, neo- antigen peptide vaccines combined with a TLR7 agonist and either anti-PD1 or anti-CD40. Aim 2: Develop novel combinations of immunization and systemic immunomodulation with improved efficacy using murine CRC tumor vaccine models. These studies will provide critical information that will guide the future of cancer vaccine design and allow for testing of vaccination efficacy in a setting of low volume disease. The approach promises to make a significant positive impact in a disease setting that shows high likelihood of recurrence and limited treatment options. Furthermore, these studies have a strong potential to make an impact in many other cancer types.